Bottom Line:
Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo.Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45.These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

ABSTRACTAvailability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Fig5: USP2-45 binds to α2δ-1 subunits. Pull-down experiments were performed on HEK-293 cells using biotinylated S-protein which recognizes an S-tag epitope inserted into USP2-45. Western blots show that USP2-45 co-precipitated with α2δ-1 but not Cav1.2 nor β2 subunits (n = 3)

Mentions:
In support of a direct effect of USP2 on the channels, we found that USP2-45 co-immunoprecipitated with α2δ-1 but not with Cav1.2 subunits (Fig. 5). As expected, we found no evidence for interaction of USP2-45 with β2 subunits (Fig. 5) which are not targeted by this de-ubiquitylase (Fig. 4a, c). These results suggest that USP2-45 binds only to the auxiliary subunit α2δ-1 and that this interaction is sufficient to promote the de-ubiquitylation of both α2δ-1 and Cav1.2. To confirm the involvement of α2δ-1, we assessed the effect of USP2-45 on channels expressed without α2δ-1 or β subunits. We show that, in the absence of α2δ-1, USP2-45 did not alter Cav1.2/β2 current densities (reduced by 9 ± 16 % at +10 mV, n = 19; NS; Fig. 6a), nor the current-voltage relationship: V50,act for Cav1.2/β2 channels was not significantly different between control (0.8 ± 1.0 mV, n = 11) and USP2-45-transfected cells (−2.6 ± 0.9 mV, n = 9; NS). By contrast, USP2-45 still reduced Cav1.2/α2δ-1 current densities in the absence of β (by 56 ± 19 % at +20 mV, n = 9; p < 0.05; Fig. 6b), again without altering the current-voltage relationship (V50,act = 6.8 ± 1.6 mV, n = 6 in control, versus V50,act = 7.7 ± 4.6 mV, n = 3; NS; in USP2-45-transfected cells), confirming that, unlike α2δ-1, β subunits are not required for regulation of Cav channels by USP2-45. Altogether, these data demonstrate the paramount role of α2δ-1 in USP2-45-induced downregulation of Cav channels.Fig. 5

Fig5: USP2-45 binds to α2δ-1 subunits. Pull-down experiments were performed on HEK-293 cells using biotinylated S-protein which recognizes an S-tag epitope inserted into USP2-45. Western blots show that USP2-45 co-precipitated with α2δ-1 but not Cav1.2 nor β2 subunits (n = 3)

Mentions:
In support of a direct effect of USP2 on the channels, we found that USP2-45 co-immunoprecipitated with α2δ-1 but not with Cav1.2 subunits (Fig. 5). As expected, we found no evidence for interaction of USP2-45 with β2 subunits (Fig. 5) which are not targeted by this de-ubiquitylase (Fig. 4a, c). These results suggest that USP2-45 binds only to the auxiliary subunit α2δ-1 and that this interaction is sufficient to promote the de-ubiquitylation of both α2δ-1 and Cav1.2. To confirm the involvement of α2δ-1, we assessed the effect of USP2-45 on channels expressed without α2δ-1 or β subunits. We show that, in the absence of α2δ-1, USP2-45 did not alter Cav1.2/β2 current densities (reduced by 9 ± 16 % at +10 mV, n = 19; NS; Fig. 6a), nor the current-voltage relationship: V50,act for Cav1.2/β2 channels was not significantly different between control (0.8 ± 1.0 mV, n = 11) and USP2-45-transfected cells (−2.6 ± 0.9 mV, n = 9; NS). By contrast, USP2-45 still reduced Cav1.2/α2δ-1 current densities in the absence of β (by 56 ± 19 % at +20 mV, n = 9; p < 0.05; Fig. 6b), again without altering the current-voltage relationship (V50,act = 6.8 ± 1.6 mV, n = 6 in control, versus V50,act = 7.7 ± 4.6 mV, n = 3; NS; in USP2-45-transfected cells), confirming that, unlike α2δ-1, β subunits are not required for regulation of Cav channels by USP2-45. Altogether, these data demonstrate the paramount role of α2δ-1 in USP2-45-induced downregulation of Cav channels.Fig. 5

Bottom Line:
Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo.Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45.These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

ABSTRACTAvailability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.